Received: 9 July 1997 / Uploaded: 21 July 1997

Introduction

Digitalis cardiac glycosides are well known drugs clinically used for treatment of congestive heart failure.¹ Their action is mainly due to inhibition of Na+,K+-ATPase, an enzyme located in the cell membrane and promoting the outward transport of Na+ and the inward transport of K+.2 Recently the existence of endogenous digitalis-like factors that may be responsible for essential hypertension3 has opened a new field in the study of compounds acting on the Na+,K+-ATPase. The most potent inhibitors of Na+,K+-ATPase are cardenolides such as digitoxigenin (Figure 1) with the following structural characteristics: 17b-unsaturated lactone, 3b- and 14b-hydroxy substituents and A/B and C/D cis ring junctions. The 14b-hydroxy group is involved in a hydrogen bonding with the receptor and plays an important role in binding digitalis compounds to Na+,K+-ATPase receptor; in fact compounds in which this group is absent show very low binding affinity or no affinity at all.4 However the known derivatives with a 14b,15b-epoxy group (Figure 1) show high binding affinities although not as high as the 14b-hydroxy analogues (Table 1).

Figure 1. Known compounds synthesized using the reported procedures.

Herein, we report the synthesis and biological evaluation of unknown 14b-methoxy derivatives of digitoxygenin and of other digitalis-like compounds. These compounds have a 14b-oxygen, which can be a hydrogen bonding acceptor, as is the case of 14b,15b-epoxide derivatives, but not a hydrogen bonding donor as is the case of 14b-hydroxy derivatives. Comparison of the binding values of these three classes of compounds could allow more insight into the requirements necessary for recognition by the receptor. Only a 3b-glucoside derivative of 14b-methoxydigitoxygenin has been described;10 for which the inotropic activity was reported to be marginal, but no synthetic route was given.

Chemistry

Attemps to introduce a methyl on the 14b-hydroxy group of digitoxigenin, with the secondary 3b-hydroxy protected, using diazomethane or dimethyl sulfate failed; diazomethane failed also when applied on the 17b-(3-furyl) analogue, while dimethyl sulfate gave low yield.
We then turned our attention to a Williamson reaction with MeI and, since the strongly basic reaction conditions proved incompatible with the presence of the a,b-unsaturated lactone of digitoxigenin, we tried the reaction on the17b-furyl derivative 2 (Scheme 1).

Scheme 1. Reagents and conditions: a: tert-butyldimethylsilyl chloride, TEA, DMF, rt (90%); b: KH, dry THF, reflux; then MeI; c: n-Bu₄NF, THF, reflux (quantitative from 2).

The known 17b-(3-furyl)-5b-androstane-3b,14b-diol 111 was reacted with tert-butyldimethylsilyl chloride in DMF in the presence of triethylamine to give the protected derivative 2 (90%); this TBS derivative and KH were kept at reflux temperature for one hour in dry THF; the addition of MeI instantaneously gave the desired 14b-methoxy derivative 3a. The crude 3a was deprotected with n-Bu4 NF in THF at reflux temperature to give 3b in quantitative yield from 2.
From the 14b-methoxy derivative 3a the 14b-methoxy digetoxigenin 6b could be obtained by the oxidative/reductive procedure6 shown in Scheme 2. The crude 3a was reacted with m-chloroperbenzoic acid in CHCl₃ in the presence of AcOH and AcONa; the crude hydroxy lactone intermediates 4a and 5a were reduced with NaBH₄ in CH₂Cl₂ to give a mixture of the desired digitoxigenin derivative 6a and of the isomeric isodigitoxigenin derivative 7a in a 8:2 ratio. The two compounds were separated by flash chromatography to give 6a (49% from 2) and 7a (13% from 2) and then deprotected by acidic hydrolysis with dil. HCl in a CHCl ₃/MeOH mixture; 6b (81%) and 7b (58%).12

The 17b-(4-pyridazinyl) derivative 8a was prepared by reacting the 17b-(3-furyl) derivative 3a with NBS in THF in the presence of AcONa and then with hydrazine7 to give, after chromatographic purification, the desired 8a (24% from 2) and the N-amino lactam derivative 9a as a side product (20% from 2); 8a was deprotected with n-Bu4 NF in THF at reflux temperature (81% yield), while 9a degraded to a complex mixture under the same conditions.

Biological Data

All the synthesized compounds were evaluated, in comparison with 14b,15b-epoxy and/or 14b-hydroxy analogues, for displacement of the specific [³H]-ouabain binding13 on Na⁺,K⁺-ATPase (Table 1).